Angiogenesis or “neovascularization” is the formation of new blood vessels from the endothelial cells (EC) of preexisting blood vessels. This process involves EC migration, proliferation, and differentiation, which begins with localized breakdown of the basement membrane in the parent vessel. The EC then migrate away from the parent vessel into the interstitial extracellular matrix (ECM) to form a capillary sprout, which elongates due to continued migration and proliferation of the cells.
Angiogenesis is typically held under strict control, and under normal conditions occurs only under certain defined physiological processes. For example, angiogenesis occurs during embryogenesis, post-natal growth, wound repair, and menstruation. Uncontrolled angiogenesis, however, can result in pathogenic conditions where the developing blood vessels destroy the surrounding tissue or sustain malignancies. Such pathogenic conditions include diabetic retinopathy, psoriasis, exudative or “wet” age-related macular degeneration (“AMD”), inflammatory disorders, and most cancers. AMD in particular is a clinically important angiogenic disease. This condition is characterized by choroidal neovascularization in one or both eyes in aging individuals, and is the major cause of blindness in industrialized countries.
Two key regulators of angiogenesis are angiopoietin-1 (“Ang1”) and angiopoietin-2 (“Ang2”). These regulators can act in concert with vascular endothelial growth factor (“VEGF”) to regulate angiogenesis, although inhibition of Ang1 or Ang2 alone appears to block neovascularization. Ang1, Ang2 and VEGF exert their effect on EC through the two VEGF receptors and another tyrosine kinase receptor called “tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2” or “Tie2.” Hackett et al. (2002), J. Cell. Phys. 192: 182-187. Whereas VEGF binding to its receptors is crucial for initiating the angiogenic process, Ang1 and Ang2 bind to Tie2 and modulate maturation of the new blood vessels. Ang1 and Ang2 are also involved in maintaining endothelial cell integrity. Lobov et al. (2002), Proc. Nat. Acad. Sci. USA 99: 11205-11210. As discussed below, agents which bind to and block the Tie2 receptor can also inhibit angiogenesis.
Ang1 and Ang2 are differentially expressed, and early studies indicated that Ang1 promoted neovascularization and Ang2 was an angiogenesis antagonist. However, evidence now shows that Ang2 can increase blood vessel diameter and promote remodeling of the basal lamina. Ang2 also appears to induce EC proliferation, migration and sprouting of blood vessels in the presence of VEGF. Lobov et al., 2002, supra.
Ang1 reportedly promotes angiogenesis during embryonic development, in particular through the modulation of endothelial-stromal cell communication and by regulating the maturation and stability of blood vessels. Lin P et al., Proc. Nat. Acad. Sci. USA 95: 8829-8834 (1998). However, the widespread expression of Ang1 and Tie2 in vascular endothelium, and phosphorylation of Tie2 in quiescent adult vasculature also suggest that Ang1 is involved in postnatal angiogenesis. Takagi et al. (2003), Inv. Ophthalm. Vis. Sci. 44: 393-402.
In contrast to the more extensive expression patterns of Ang1 and Tie2, Ang2 appears to be expressed only at sites of vascular remodeling. Takagi et al. (2003), supra. For example, Ang2 expression is markedly increased in ovary, uterus and placenta during menstruation. Ang2 expression levels also follow a cyclical pattern of expression in the corpus luteum, which parallels the cycle of quiescence, angiogenesis and vascular regression of this structure (i.e., Ang2 levels are low during quiescence and high during angiogenesis and regression). Hackett et al., 2002, supra. Ang2 is also induced by hypoxic cytokines, including VEGF, and is expressed in tissues undergoing pathologic angiogenesis associated with tumors, AMD and in an animal model of retinal ischemia. Takagi et al., 2003, supra. Moreover, Ang2 is upregulated in the epiretinal membranes of patients with ischemic retinal disorders, but not in membranes from patients with non-ischemic retinal disorders. The expression of Ang1, however, remains similar in epiretinal membranes from patients with ischemic or non-ischemic disorders. Takagi et al., 2003, supra.
Ang2 and Tie2 are co-localized in the EC of highly vascularized regions, and Tie2 is overexpressed in areas of vascular remodeling. Asahara T. et al., Circ. Res. 83: 223-240 report that Ang1 and Ang2 have similar synergistic effects with VEGF to promote angiogenesis in a mouse corneal neovascularization assay. Thus, Ang1, Ang2 and Tie2 play an important role in both normal and pathogenic neovascularization in developing and adult organisms.
Ang1, Ang2 or Tie2 are therefore attractive therapeutic targets for treatment of pathogenic angiogenesis. For example, Lin P et al. (1998), supra, inhibited tumor growth and metastasis in a mouse model by expressing a soluble recombinant Tie2 receptor. The recombinant Tie2 protein blocked ligand binding to endogenous Tie2 receptors, but likely produced only a stoichiometric reduction in Ang2/Tie2 binding. Takagi et al., 2003, supra inhibited of Tie2 signaling with a soluble fusion protein containing the ectoplasmic domain of Tie2, which suppressed hypoxia-induced retinal angiogenesis both in vitro and in vivo. Asahara et al. (1998), supra showed that administration of a soluble Tie2 receptor abolished the effects of Ang1 or Ang2 on VEGF-induced neovascularization in the mouse cornea. However, therapeutic strategies based on agents such as soluble Tie2 receptors are not preferred, however, because such agents would likely be overwhelmed by the high production of Ang2 or Tie2 in the EC of highly vascularized areas.
RNA interference (hereinafter “RNAi”) is a method of post-transcriptional gene regulation that is conserved throughout many eukaryotic organisms. RNAi is induced by short (i.e., <30 nucleotide) double stranded RNA (“dsRNA”) molecules which are present in the cell (Fire A et al. (1998), Nature 391: 806-811). These short dsRNA molecules, called “short interfering RNA” or “siRNA,” cause the destruction of messenger RNAs (“mRNAs”) which share sequence homology with the siRNA to within one nucleotide resolution (Elbashir S M et al. (2001), Genes Dev, 15: 188-200). It is believed that the siRNA and the targeted mRNA bind to an “RNA-induced silencing complex” or “RISC”, which cleaves the targeted mRNA. The siRNA is apparently recycled much like a multiple-turnover enzyme, with 1 siRNA molecule capable of inducing cleavage of approximately 1000 mRNA molecules. siRNA-mediated RNAi degradation of an mRNA is therefore more effective than currently available technologies for inhibiting expression of a target gene.
Elbashir S M et al. (2001), supra, has shown that synthetic siRNA of 21 and 22 nucleotides in length, and which have short 3′ overhangs, are able to induce RNAi of target mRNA in a Drosophila cell lysate. Cultured mammalian cells also exhibit RNAi degradation with synthetic siRNA (Elbashir S M et al. (2001) Nature, 411: 494-498), and RNAi degradation induced by synthetic siRNA has recently been shown in living mice (McCaffrey A P et al. (2002), Nature, 418: 38-39; Xia H et al. (2002), Nat. Biotech. 20: 1006-1010). The therapeutic potential of siRNA-induced RNAi degradation has been demonstrated in several recent in vitro studies, including the siRNA-directed inhibition of HIV-1 infection (Novina C D et al. (2002), Nat. Med. 8: 681-686) and reduction of neurotoxic polyglutamine disease protein expression (Xia H et al. (2002), supra).
What is needed, therefore, are agents and methods which selectively inhibit expression of Ang1, Ang2 or Tie2 in catalytic or sub-stoichiometric amounts, in order to effectively decrease or block angiogenesis.